Human respiratory Syncytial virus is classified in the genus Pneumovirus, family Paramyxoviruses. It is a major cause of severe lower respiratory tract disease in infants, the elderly and in immunocompromised individuals. It is also an important factor in upper respiratory tract disease in older children and adults. Currently there is no effective h-RSV vaccine available in the art.
RSV is an enveloped RNA virus that expresses two major antigens at its surface: the attachment protein G and the fusion protein F. Both proteins appear to invoke protective antibodies. G is the determinant of the two known h-RSV subgroups A and B. Antigenic differences can be found within the two groups. The G protein shows a high degree of variation with only 53% amino acid homology between groups A and B and up to 20% differences in G protein sequences within group A (Mufson 1988, Cane 1991).
Passive immunisation with RSV-enriched immunoglobulin (Respigam) or synthetic humanised monoclonal antibodies against F (Palivizumab) is currently used to treat and protect neonates of certain predispositions (e.g. premature birth) against RSV infection (Robinson 2000, Greenough 2000). RSV pathology has two major aspects: cell damage caused by the virus itself and tissue damage caused by the overreacting immune system. The latter is a highly complicating factor in vaccine design.
RSV infections are seasonal, limited to the winter period and peak in the Northern Hemisphere around the end of the year. RSV infects every child before the age of two, in many cases twice. Older individuals on average are infected every other year, depending on the setting; people in close contact with infants and young children have a 50% risk. The virus spreads by close contact, in droplets or through contaminated surfaces. RSV is not efficiently spread through aerosols; the virus particles are relatively unstable. Internal spread of the virus from the upper respiratory tract (URT) to the lower respiratory tract (LRT) occurs predominantly by inhalation of virus particles produced in the URT epithelium during primary infection. Spread through syncytium formation (one of the pathological properties of the virus, which gave it its name) can not be ruled out and may play a secondary role in LRT infection.
In general, RSV pathology starts in the URT; the port of entry is the nose and to a lesser extent the eyes—not the mouth. When restricted to URT tissues, disease is limited to common cold, although in adults sometimes severe. However, when the virus can reach the LRT, bronchiolitis and pneumonia can ensue in unprotected individuals. In young infants, this can be life threatening, approx. 1/100 will require hospitalisation and mechanical ventilation, out of these 1% may die. In the elderly, RSV-induced LRT disease is a major cause of hospitalisation; it is suspected that RSV causes 25% of flu-like diseases.
The immune response to RSV is complex. In general, exposure to h-RSV will build up a response that protects against LRT disease. This response wanes with older age, causing the higher susceptibility to RSV of the older population. Effective long lasting protection against URT disease appears not feasible: re-infection is very common, even within the same season and this is not caused by viral variation. Protection against RSV infection involves antibodies against viral proteins F and G circulating in the blood, which can prevent LRT disease. URT infection can be controlled by mucosal antibodies against F and G, but these have a limited life span. CD8+ T cells against as yet unidentified viral proteins are required to clear the virus from infected tissues, but they appear to be short-lived or inefficiently recruited from their reservoirs. Most likely, this is caused by RSV-expressed factors, possibly encoded in the G gene (Srikiatkhachorn, 1997a).
An important aspect of RSV disease is immune enhancement of pathology. In limited cases the cellular immune response can exacerbate RSV disease by the action of cytokines on infected tissues released from excessively attracted granulocytes. Host predisposition is involved in this reaction, but possibly also the timing of first RSV infection after birth. Unfortunately, early vaccine trials with formalin-inactivated RSV showed that in these vaccination settings immune enhanced pathology upon wt infection was prevalent (Kim 1969). Factors contained in RSV appear to be responsible for this phenomenon and were apparently released by formalin treatment. In the 40 years since then, it was gradually shown that the viral G protein is the predominant mediator of these problems, but the mechanism remains unclear (Srikiatkhachorn 1997b). In any case, vaccination with a G protein out of the context of the virion (i.e. in inactivated virus preparations, as expression product not properly embedded in a membrane or in the form of peptides) seems to be causing immune enhancement in model systems. Thus, although G contributes to some extent to RSV immunity, its properties also complicate vaccine design.
Initial live RSV vaccine candidates included cold passaged or temperature-sensitive mutants. The former have been attenuated by culturing at decreasing temperature, leading to dependency on low temperatures for growth, whereas the latter mutants have been made dependent on a specific, usually higher temperature for replication by chemical or radiation mutagenesis. These live virus vaccine candidates appeared to be either under- or overattenuated (Crowe 1998).
Subunit vaccine candidates are derived from either the RSV-F or the G protein, being the main targets for neutralising antibodies. A candidate subunit vaccine, PFP2, purified F protein, is safe in RSV-seropositive patients, but it did not provide full protection against LRT infection and associated disease (Gonzalez 2000). Another subunit vaccine approach is BBG2Na, which consists of a polypeptide, comprising amino acid 130-230 of h-RSV-G, fused to the albumin-binding domain of streptococcal G protein (Power 1997). BBG2Na induces a T helper type 2 response in neonatal mice, and does not elicit lung immunopathology (Siegrist 1999). There is no data yet on protection. The use of new adjuvants for a balanced humoral and cellular immune response are currently under investigation in animal models (Plotnicky 2003).
The use of plasmid-DNA vectors encoding RSV-F and G antigens as vaccine candidates has been studied in animal models. These vaccines induce protective responses in rodents (Li 2000), but in one study RSV-F DNA vaccine candidate immunised mice developed a slightly enhanced pulmonary inflammatory response following challenge with wt virus (Bembridge 2000). The feasibility of the use of plasmid DNA vaccines in humans is not yet known and it will likely take at least 15 years before this approach is sufficiently studied and—more importantly—accepted, particularly for neonates. Candidate vaccines based on vector delivery systems are constructed of live recombinant vectors expressing RSV proteins. For example, recombinant vaccinia virus expressing RSV-F and G provided protection in mice, but lacked this effect in chimpanzees (Collins 1990). The question is whether these systems are safe (notably vaccinia virus) and feasible in the light of existing (maternal) antibodies against poxviruses in the community and the main target group being neonates.
Several vaccine candidates are based on recombinant live RSV, generated by reverse genetics. One line of study focuses on attenuating these viruses by introducing the individual or combined mutations responsible for cold-adaptation and temperature-sensitivity into the recombinant virus. None of these vaccine candidates were usable, because of either over- or underattenuation. Another line of study focuses on deletion of one or more viral non-structural genes. Limited data are available on the behaviour of these viruses in model systems (Jin 2003).
An alternative approach to RSV vaccine development is the use of bovine RSV. A chimeric bovine RSV with either the human F protein alone or both the human F and G protein was evaluated for its efficacy in chimpanzees. This vaccine candidate was restricted in replication to such a degree that animals were not protected after wild type h-RSV challenge (Buchholtz 2000).
Thus, currently there is no effective h-RSV vaccine available in the art. All RSV vaccine candidates that have been tested in animal models are unusable in humans. There is thus a long felt need in the art for RSV vaccines that are both effective and safe and it is an object of the present invention to provide for such vaccines.